1. Field of the Invention
The present invention relates to a reaction method with use of a biochemical analysis unit which is used for base sequence analysis of DNA and the like.
2. Description Related to the Prior Art
In order to make a biochemical analysis for base sequence of substances derived from living organism (for example DNA), a biochemical analysis unit is used. In order to obtain the biochemical analysis unit, minute through holes are formed in the substrate, and porous materials and the like are pressed into each through hole to form a spot area. Thus, the spot areas are arranged on the substrate, and therefore the biochemical analysis unit is called also microarray. A method of biochemical analysis, in which the biochemical analysis unit is used, includes a spotting process, a reaction process, a data reading process, and a data analysis process. In the spotting process, a specific binding substance as a reagent (hereinafter probe) is spotted and fixed in the spot areas on the biochemical analysis unit. In the reaction process, a specific binding substance as a test body (hereinafter target) is penetrated into the spot areas, and the specific binding (the biding between the probe and the target) is made. In the data reading process a biochemical analysis data is read out from the biochemical analysis unit as a result of the specific binding reaction in each spot area. In the data analysis process, the read out analysis data is analyzed in the personal computer and the like. (see, Japanese Patent Laid-Open Publication No.2002-355036).
Since the probe is a reagent for searching the information of expression, the molecular structure (for example base sequence, composition and the like) of the used probe is already known. As the probe, there are substances derived from living organism (such as hormones, tumor markers, enzymes, antibodies, antigens, abzymes, receptors, other proteins, ligand, nucleic acids, cDNA, DNA, mRNA, and the like, which are extracted and isolated from the living organism), and products obtained by performing the chemical treatments or the chemical modifications of the substances derived from living organism.
When the base sequence is searched, several sorts of the probes are fixed in respective spot areas of the biochemical analysis unit in the spotting process. Then in the reaction process, a solution in which the target is dissolved to a solvent is penetrated in the spot areas, and the specific binding of the target and the probe having a complemental relation to the target is made. In order to detect the specific binding, the reaction solution contains for example labeling substances. As the labeling substances to be used, there are radioactive substances which generate a radial ray. After the specific binding is made, the biochemical analysis unit is cleaned to remove the reaction solution on other areas than spot areas.
In the spot area in which the specific binding is made, the labeling substances remain. Thus the specific binding is detected based on the radial ray from the labeling substance in the data reading process. As a device for reading data, a scanner is used. However, in an image pickup apparatus for reading the optical information, the radial image cannot be directly picked up. Accordingly, when the radioactive substances are used as the labeling substances, a stimulable phosphor sheet is used. In the stimulable phosphor sheet, the radioactive energy in the spot area in which the specific binding is made is stimulated and generates a light as a converted optical information in accordance with the stimulated energy. In the stimulable phosphor sheet are formed stimulable phosphor areas (areas containing stimulable phosphors) to be exposed to the radial ray. When the stimulable phosphor areas are overlapped with the reacted spot areas (the spot areas in which the specific binding reaction is made), the exposure of the some stumilable phosphor areas is made in radiation of the radial rays generated from the reacted spot areas. When an exiting light is illuminated on the some stimulable phosphor areas after the exposure, lights are generated from the some stimulable phosphor areas to form an image as a biochemical analysis data which is to be read.
In order to make the specific binding reaction, the shaking is usually made in the prior art. In the shaking method, a specific binding reaction solution (hereinafter reaction solution) and a biochemical analysis unit in which the probes are fixed are set in a reaction vessel, and the reaction vessel is shaken on a shaking stage. Thus the reaction solution containing the target is applied to and then penetrates into the spot areas. However, in the shaking method, as the examiner exchanges the reaction solution with his hands, there are following problems. Firstly, the equivalent penetration of the reaction solution is hard among the spot areas. Secondly, the penetration pressure into the spot areas is low, and the reaction is often made slowly, for example, at least for more than 10 hours, and for few days when it takes long time. Thirdly, not only for the different but also for the same examiner, it is hard to keep the supplied and the discharged reaction solution to the same temperature. Thus the experimental conditions change and the experiment cannot be replicated correctly.
Accordingly, instead of the shaking method, a flow through method is applied to the reaction method (See, WO 01/45843), in which a mechanical pressure of such as a piston, a pump and the like drives to flow the reaction solution from one side to another side in the spot area.
The reaction vessel to be used in the flow through method has a reaction chamber (hereinafter a chamber) in which the biochemical analysis unit is contained and the reaction solution is supplied. The chamber is provided with an inlet through which the reaction solution is supplied, and an outlet through which the reaction solution after flowing through the spot areas is discharged. The inlet and outlet are positioned so as to confront to a middle portion of the flow-through area in which the plural spot areas are arranged. The pump and the piston are driven to mechanically increase the pressure for feeding the reaction solution into the chamber. Thus the reaction solution flows into the chamber with increased feeding pressure, the pressure of the reaction solution for penetrating into the spot areas becomes more, and the reaction speed is increased. Further, with the mechanical drive of the piston, the pump and the like, the feed pressure is kept constant. Therefore the experimental condition does not become different.
On walls of the chamber is formed a taper which is curved towards the biochemical analysis unit such that the specific binding reaction solution supplied through the inlet can equivalently flow through all over the flow-through area.
However, in accordance with downsizing the chamber, the formation of the taper becomes difficult and the cost increases. Further, several steps of the reactions are sometimes made with use of the plural kinds of reaction solutions and the cleaning solution. In this case, since the physical properties of those solutions may change, it becomes hard that the reaction solution or the cleaning solution always equivalently flow through all over the flow-through area.